The single Fe(II) in reduced rubredoxin from Clostridium pasteurianum was found to be quantitatively displaced by either Cd2+ or Zn2+ when a modest molar excess of the substituting metal salt was anaerobically incubated with the reduced rubredoxin under mild conditions, namely, room temperature, pH 5.4-8.4, and no protein denaturants. Under the same conditions, cadmium-for-zinc substitution was also achieved upon aerobic incubation of the zinc-substituted rubredoxin with a modest molar excess of Cd2+. Displacements of Fe(II) from the reduced rubredoxin were not observed upon anaerobic incubation with Ni2+, Co2+, or VO2+ salts, and no reaction with any of the divalent metal ions was observed for the oxidized [Fe(III)] rubredoxin. Fe(II) could not be re-inserted into the Zn- or Cd-substituted rubredoxins without resorting to protein denaturation. H-1 and Cd-113 NMR experiments showed that the cadmium-substituted rubredoxin prepared by the non-denaturing substitution method retained the pseudotetrahedral M(SCys)(4) coordination geometry and secondary structural elements characteristic of the native rubredoxin, and that "unzipping" of the beta-sheet did not occur during metal substitution. Rates of Fe(II) displacement by M2+ (M = Cd or Zn) increased with increasing M2+/rubredoxin ratio, decreasing pH, and lower ionic strength. The substitution rates were faster for M = Cd than for M = Zn. Rates of Cd2+ substitution into a V8A-mutated rubredoxin were significantly faster than for the wild-type protein. The side-chain of Vg is on the protein surface and close to the metal-ligating Cys42S gamma at the M(SCys)(4) site. Therefore, the rate-limiting step in the substitution process is suggested to involve direct attack of the [M(SCys)(4)](2-) site by the incoming M2+, without global unfolding of the protein. Implications of these results for metal ion incorporation into rubredoxins in vivo are discussed.
Direct metal ion substitution at the [M(SCys)(4)](2-) site of rubredoxin / F. Bonomi, S. Iametti, D.M. Kurtz, E.M. Ragg, K.A. Richie. - In: JBIC. - ISSN 0949-8257. - 3:6(1998 Dec), pp. 595-605.
|Titolo:||Direct metal ion substitution at the [M(SCys)(4)](2-) site of rubredoxin|
BONOMI, FRANCESCO (Primo)
IAMETTI, STEFANIA (Secondo)
RAGG, ENZIO MARIA (Penultimo)
|Parole Chiave:||Cadmium; Iron-sulfur proteins; Metal substitution; Rubredoxin; Zinc|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
Settore CHIM/06 - Chimica Organica
|Data di pubblicazione:||dic-1998|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1007/s007750050272|
|Appare nelle tipologie:||01 - Articolo su periodico|